1. Field of the Invention
The present invention relates generally to test articles and assays for the detection of analytes in biological fluid samples. More particularly, the present invention relates to test articles and assays which employ dyed microorganisms as visual labels to detect the suspected analytes.
The use of chromogenic and fluorescent dyes as "labels" in biological assay procedures is known. Typical assay protocols call for direct or indirect binding of a dye label to an analyte or analyte analog in a biological sample, where the presence or absence of the dye at a particular stage of the assay can be determined visually and related to the amount of analyte initially present in the sample. A wide variety of specific assay protocols exist.
Of particular interest to the present invention, certain assays utilize naturally colored or dyed particles as a label, where the particles are bound to an antibody or other specific binding substance. Suggested particles include dyed latex beads, dye imbibed liposomes, erythrocytes, metal sols, and the like. The colored particle in such complexes can serve as a visible marker, where separation, capture, or aggregation of the particles is mediated through binding of the antibody or other specific binding substance. The amount of label thus segregated in a particular assay step is related to the amount of analyte initially present in the sample.
A variety of methods for preparing such antibody-particle compositions have been proposed. Such methods generally rely on producing a colored particle, typically by dying a latex bead, a liposome, or the like, and subsequently attaching the colored particle to the antibody, typically by passive adsorption or by covalent binding.
While generally useful, methods for preparing such antibody-particle compositions can be relatively complex, usually requiring multi-stage operations including preparation of the particle, coloring of the particle, attachment of the particle to the antibody, and blocking of the particle for use in immunoassays. Some particles, such as liposomes, are relatively unstable and do not provide uniform characteristics following storage or during use in some samples. Moreover, a loss of antibody binding capacity can often result from the particle attachment. Sometimes these particles are not compatible with the antibodies selected for a particular application. Often times the antibodies are bound to the particle inefficiently and without regard to orientation for maximal immunological reactivity.
It would be desirable to provide improved labeling compositions that would allow for antibodies of any specificity to be used for an application. The compositions should be relatively easy to prepare, with a reduced cost, and have uniform characteristics. In particular, the compositions should retain antibody activity to a significant extent with proper antibody orientation, i.e. Fc region bound, and be very stable during storage and under assay conditions to provide for long product shelf life. It will be appreciated, however, that the methods and compositions of the present invention need not be superior to the prior art in each or any of these aspects, but rather that these are general advantages that the present invention can provide relative to certain prior art methods and products.
2. Description of the Background Art
U.S. Pat. No. 4,943,522, describes a solid phase lateral flow assay using erythrocytes as a label. U.S. Pat. No. 4,863,875, describes compositions comprising at least ten dye molecules or monomers covalently attached to an antibody through an isocyanate group on the dye. U.S. Pat. No. 4,861,711, describes a solid phase lateral flow assay using enzyme antibody conjugate and substrate, each separately held in absorbent pads. U.S. Pat. No. 4,703,017, describes a solid phase assay device which relies on specific binding of a ligand-label conjugate on a solid support, where the label is disclosed as a particle, such as, i.e., a liposome, or polymer microcapsule. U.S. Pat. No. 4,608,246 describes assays for typing blood which employ erythrocytes as a labelling agent. U.S. Pat. No. 4,452,886, describes the covalent attachment of photon absorbing or emitting polymers to proteins, such as antibodies and antigens. U.S. Pat. No. 4,373,932, describes labeling of a ligand with an aqueous dispersion of a hydrophobic dye or pigment, or a polymer nuclei coated with such a dye or pigment. U.S. Pat. No. 4,313,734 describes methods of detecting sample analytes by the determination of the metallic label content in the sample. U.S. Pat. No. 4,169,138 describes immunoassays which employ visible particles including undyed microorganisms, bound to polymers which may be of microbial origin. See also, U.K. Patent No. 2,204,398; EP Patent No. 306 722; and EP Patent No. 276 152, which relate to lateral flow assays.
Enzyme assays and immunohistochemical staining techniques which produce a colored dye by reaction of a substrate with an enzyme bound to a target moiety are known. Jonsson et al., J. Immunol., 4:29-33, describes a radioimmunoassay employing Staphylococcus aureus bacteria as a solid phase for separation of IgG-antigen complexes from free antigen. Leuvering and Van de Waart, J. Immunoassay, 1:77-91, describes immunoassays employing inorganic sol particles as labels. Guesdon and Avraneas, J. Immunol. Meth., 39:1-13, and Prenot and Guesdon, Ann. Virol., 132:529-542, describe immunoassays employing erythrocytes as the labelling composition.